Bioorganic & Medicinal Chemistry 21 (2013) 2229–2240

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Bioorganic & Medicinal Chemistry

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Discovery of thienopyrimidine-based inhibitors of the humanfarnesyl pyrophosphate synthase—Parallel synthesis of analogsvia a trimethylsilyl ylidene intermediate

0968-0896/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.bmc.2013.02.006

⇑ Corresponding author. Tel.: +1 514 398 3638; fax: +1 514 398 3797.E-mail address: youla.tsantrizos@mcgill.ca (Y.S. Tsantrizos).

PO(OH)2(HO)2OP4

2N N

H

PO

PO3

O

N

N

S

R5

R6

NH2

1N

N

S

R5

R6

HN

PO(OH)2

PO(OH)2

Figure 1. Structures of thieno[2,3-d]pyrimid-4-amines (general structurenopyrimidine-based bisphosphonate inhibitors of hFPPS/hGGPPS (general s2), hFPPS inhibitor 3 and hGGPPS inhibitor 4.

Chun-Yuen Leung a, Adrienne M. Langille a, John Mancuso a,c, Youla S. Tsantrizos a,b,⇑a Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, QC, Canada H3A 0B8b Department of Biochemistry, McGill University, 3649 Promenade Sir William Osler, Montreal, QC, Canada H3G 0B1c Tranzyme Pharma, 3001 12th Avenue North, Sherbrooke, QC, Canada J1H 5N4

a r t i c l e i n f o

Article history:Received 4 January 2013Revised 1 February 2013Accepted 11 February 2013Available online 18 February 2013

Keywords:Thienopyrimidine bisphosphonatesModified Gewald synthesis2-(1-(Trimethylsilyl)ethylidene)malononitrileFarnesyl pyrophosphate synthase

a b s t r a c t

Thienopyrimidine-based bisphosphonates were identified as a new class of nitrogen-containingbisphosphonate (N-BP) inhibitors of the human farnesyl pyrophosphate synthase (hFPPS). Analogs wereprepared via cyclization of 2-(1-(trimethylsilyl)ethylidene)malononitrile to 2-amino-4-(trimethylsilyl)thiophene-3-carbonitrile in the presence of elemental sulfur. Direct ipso-iododesilylation of this interme-diate led to selective iodination at Cb of the sulfur atom in high efficiency. The synthetic protocols devel-oped were used in the parallel synthesis of structurally diverse thieno[2,3-d]pyrimidin-4-amine-basedbisphosphonate inhibitors of hFPPS.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction potential inhibitors of the human farnesyl pyrophosphate synthase

(OH)2

(OH)2

The thienopyrimidine core is an integral part of numerousbiologically active compounds, as evidenced by the plethora ofliterature and industrial patents disclosing the use of this heterocy-cle in agrochemicals1–3 and human therapeutics (e.g., antifungal4

and antiviral5 agents). Thienopyrimidines are often used asbioisosteres of purine nucleobases (e.g., adenine),5 and have beenemployed extensively in the design of various kinase inhibitors,including Aurora kinase,6 tyrosine kinases,7,8 cyclin-dependentkinase,9 c-Jun N-terminal kinases,10–12 PERK,13 phosphoinositide3-kinase a (PI3Ka),14,15 and EGFR/ErbB-2.16 Many of these inhibi-tors are currently under pre-clinical or clinical investigations forthe treatment of cancer, inflammatory, autoimmune and neurode-generative diseases. Thienopyrimidine-based compounds havealso been reported as inhibitors of c-secretase17 and antagonistsof the adenosine A2A receptor,18–20 and implicated in the potentialtreatment or prevention of Alzheimer’s disease.

In our own investigations, we focus on the identification of struc-turally diverse molecules that selectively inhibit mammalian prenylsynthase enzymes. Inspired by the privileged biopharmaceuticalproperties of the thienopyrimidine scaffold (1; Fig. 1), we decidedto explore thienopyrimidine-based bisphosphonates (2; Fig. 1), as

(hFPPS) and/or geranylgeranyl pyrophosphate synthase (hGGPPS);these prenyl synthase enzymes are functionally closely related.

The development of synthetic methodologies amenable toparallel synthesis of structurally diverse libraries of thienopyrimi-

1), thie-tructure

R4

O CNNC

R4

S8, Base

S

CNR4

NH2R5

NC CN

5a, R4 = (CH2)2CH35c, R4 = p-NO2-C6H4

67a, R4 = (CH2)2CH3, R5 = H7b, R4 = CH3, R5 = CH2CH37c, R4 = p-NO2-C6H4, R5 = H

7d, R4 = R5 = HS

SHO

OH

NC CN

8N

NH

O

S

2) Zn, AcOH

1) Br2, AcONa

HCO2H,conc. H2SO4

reflux

uW 100 oC

H2O

N

NH

O

S

Br

N

N

R4

S

Br

2) I2N

N

Cl

S

I1) i-PrMgBr, 0 oC

NH4OH90 oC

11a, R4 = Cl

11b, R4 = NH2

9

1012

POCl3

uW, 100 oC

Scheme 1. Gewald synthesis of 2-aminothiophene-3-carbonitriles (7) and halo-substituted thienopyrimidines.

2230 C.-Y. Leung et al. / Bioorg. Med. Chem. 21 (2013) 2229–2240

dine bisphosphonates (2) were required to support our medicinalchemistry efforts. We anticipated that the final compounds couldbe made from highly substituted thieno[2,3-d]pyrimidin-4-amineintermediates (1), after coupling of the exocyclic amine (at C-4)with the bisphosphonate moiety; the latter step could be easilyachieved using previously developed procedures for the prepara-tion of 2-aminopyridine bisphosphonates (e.g., hFPPS inhibitor 3;Fig. 1).21,22

Traditionally, thieno[2,3-d]pyrimid-4-amines (1) have beensynthesized via a multistep process from 2-aminothiophenes (7;Scheme 1). However, preparation of highly substituted thienopyr-imidine libraries is usually hampered by limitations in selectivelyfunctionalizing the Ca and/or Cb positions of the thiophene core(7). Similarly, direct and selective substitution at those positionsin the corresponding thieno[2,3-d]pyrimidine is very challenging(i.e., the C-5 and C-6 of 1).

The 2-aminothiophene core (7) can be easily prepared by theclassical Gewald method (Scheme 1).23 One-step and two-stepprocedures, under either thermal or microwave conditions, havebeen reported with various optimizations for the condensation ofthe Knoevenagel ylidene 6 with elemental sulfur to give the thio-phene 7. The drawbacks to this elegant protocol can be regioselec-tivity in the cyclization step, when non-symmetrical ketones (5)are used with an alkyl or a benzylic substituent, and the chemicalstability of ylidene 6. For example, the one-pot condensation ofpentane-2-one (5a) with malononitrile, sulfur and catalyticamounts of imidazole, was recently reported to produce a mixtureof thiophenes 7a and 7b in 1:3.4 ratio and in modest yield (Scheme1).24 Furthermore, acetophenones bearing electron withdrawingsubstituents on the phenyl ring (e.g., 5c) often produce very lowyields of the desired 2-aminothiophene (7c) under standard Ge-wald reaction conditions.25 The low yields have been attributed,in part, to decomposition and dimerization of ylidene 6, under hightemperatures and basic conditions.25

Alternatively, the unsubstituted 2-amino-thiophene-3-carboni-trile core (7d) has also been prepared from 2,5-dihydroxy-1,4-dithiane (8) and subsequently elaborated to substituted thie-no[2,3-d]pyrimidines.26,27 Chlorination or bromination at Ca ofthe thiophene sulfur can be easily achieved with NCS or NBS,respectively. However, direct and regiospecific halogenation atthe b-carbon, or the equivalent carbon on any thiophene-contain-

ing bicyclic heterocycle, is unknown. Recent reports on the prepa-ration of 3-bromothiophenes usually describe the generation of thea-lithium-b-bromothiophene from the 2-bromothiophene usingthe (so-called) base-catalyzed halogen dance (BCHD) reaction.28–32 This approach has also been used in the synthesis of4-chloro-5-iodothieno[2,3-d]pyrimidine (12) from the LDA-medi-ated BCHD rearrangement of the 6-bromo-4-chlorothieno[2,3-d]pyrimidine first to the 5-bromo analog 11a, followed bytreatment with a Grignard reagent and quenching of the anionwith iodine (Scheme 1).1,3 Several alternative approaches havebeen reported, most commonly involving 2,3-dibromination of 9,followed by reductive dehalogenation at the more reactive Ca po-sition of the thiophene with Zn in acetic acid to give intermediate10 (Scheme 1).10,11 Two additional synthetic steps are required toconvert 10–11b, which can then be used to prepare C-5 mono-substituted thienopyrimidines. Herein, we report an alternativeprotocol that allows selective disubstitution at either Ca or Cb ofthe thiophene core and is amenable to modular parallel synthesisof structurally diverse libraries of thieno[2,3-d]pyrimidin-4-amines (1). We also provide preliminary biological data whichindicates that the thienopyrimidine-based bisphosphonates de-scribed herein are inhibitors of the human FPPS and new leadsfor drug discovery. Up-regulation of hFPPS has been linked tonumerous human disorders,33,34 including cancer35 and neurode-generative diseases, thus inhibitors of this enzyme have the poten-tial of becoming valuable human therapeutics.

2. Results

2.1. Chemistry

As part of our continued interest in developing efficient, robustmethodologies that are amenable to parallel synthesis of structur-ally diverse libraries of biologically active compounds,21,22 wemodified the classical Knoevenagel/Gewald approach by incorpo-rating the trimethylsilyl ylidine 14 as the precursor to the 2-ami-no-thiophene-3-carbonitrile scaffold (15; Scheme 2). The ylidine2-(1-(trimethylsilyl)ethylidene)malononitrile (14) was preparedin nearly quantitative yield via condensation of acetyltrimethylsi-lane (13) with malononitrile under acidic conditions (Scheme 2).Ylidine 14 was found to be stable at �20 �C for several months

Table 1Mini-library of thieno[2,3-d]pyrimidin-4-amines (1) synthesized

1a, R2 = a, R5 = R6 = H1b, R2 = R6 = H, R5 = a1c, R2 = R5 = H, R6 = Br1d, R2 = R5 = H, R6 = c1e, R2 = R5 = H, R6 = e1f, R2 = R5 = H, R6 = f1g, R2 = R5 = H, R6 = i1h, R2 = R6 = H, R5 = d

NO2 CF3

N

a c d e f

i jg h k

R2, R5 and/or R6 = H or a fragment from a to k

b

CH3

S

N

O

NN

N

N

NH2

SR6

R5

1i, R2 = R6 = H, R5 = e1j, R2 = R6 = H, R5 = f1k, R2 = H, R5 = g, R6 = a1l, R2 = H, R5 = h, R6 = a1m, R2 = H, R5 = j, R6 = a1n, R2 = H, R5 = k, R6 = a1o, R2 = H, R5 = b, R6 = a

R2

S

CNTMS

R

15, R=NH216, R = NCHNMe2

S

CNTMS

NBr

S

CNTMS

NR5

S

CNR4

NR5

N

N

NH2

S1

R6

R5

18, R5 = a-k

19a, R4 = Iodine, R5 = a-k

19b, R4 = a-k, R5 = a-k

17N(CH3)2

N(CH3)2

N(CH3)2

f

i, j

h, c

c

N

N

NH2

S

R5

20a, R5 = TMS

20b, R5 = Iodine

g

g

TMS

OCNNC

TMS

b

1314

c

a

d

g

e

N

N

HN

S2

R6

R5

PO(OH)2

PO(OH)2

k, l

Scheme 2. Modular synthesis of highly substituted thieno[2,3-d]pyrimidin-4-amines; the stuctures of substituent a–k are shown in Table 1. Reagents andconditions: (a) CH2(CN)2, NH4OAc, AcOH, C6H6, Dean–Stark trap, 95 �C, 24 h (90%);(b) S8, Et2NH, pyridine, rt, 18 h (85%); (c) HCONH2, 130 �C, 48 h (75–85%); (d) ICl,DCM, �10 �C (>98%); (e) (CH3O)2CHN(CH3)2, DMF, rt, 4 h (90%); (f) NBS, DMF, rt,13 h in the dark (80%); (g) various cross-coupling reactions under standard Suzuki,Buchwald–Hartwig, Sonogashira and Stille conditions, isolated yields varied from50% to 95%, reactions were not individually optimized (for the conversion of 20b or18 to 1, R6 = H or R5 = H, respectively); (h) TBAF, THF, 0 �C to rt, 3 h (>95%); (i)CF3CO2Ag, THF, �78 �C, 15 min; (j) I2, THF, �78 �C, 3 h (>98%); (k) CHOEt3,diethylphosphite, 130 �C, 24 h (50–75%); (l) TMSBr, MeOH, rt, 72 h (>85%).

C.-Y. Leung et al. / Bioorg. Med. Chem. 21 (2013) 2229–2240 2231

and at RT for several weeks without any evidence of decomposi-tion. Condensation of 14 with elemental sulfur in pyridineprovided the 2-amino-4-(trimethylsilyl)-thiophene-3-carbonitrile(15) as a dark orange/brown solid in >85% yield.

In order to explore direct cross-coupling reactions selectively atthe Cb of the sulfur in the thiophene core (i.e., where intermediate1 is mono-substituted at C-5), we initially focused on preparingthe iodothiophene core, as a precursor to the 5-iodothienopyrimi-dine 20b (Scheme 2). Efforts to iodinate intermediate 15 at Cb,using silver trifluoroacetate and elemental iodine,36,37 lead todecomposition of the starting material at temperatures rangingfrom 0 to �78 �C. Previous examples of ipso-iododesilylation ofthiophenes under such conditions were reported to give pooryields when the TMS was adjacent to substituents with sp charac-ter.37 Cyclization of 15 with formamide produced the 5-(trimethyl-silyl)thieno[2,3-d]pyrimidin-4-amine (20a) in 75–80% yield(Scheme 2; step c). Mindful of the facile decomposition ofthiophenes upon exposure to light, this reaction was usuallycarried out in the dark. Subsequently, we reattempted to perform

an ipso-iododesilylation reaction with silver trifluoroacetate andelemental iodine; however, this reaction resulted in mostly unre-acted starting material. Alternatively, excellent yields (>98%) of20b were obtained when 20a was treated with ICl in DCM at�10 �C and the reaction was quenched at low temperatures (below0 �C) with H2O.38,39 We noted that if the reaction mixture wasallowed to warm-up above 0 �C, the isolated yield was significantlylower and mixtures of di-halogenated side products were observedby LC–MS. The iodo product 20b was found to be a useful buildingblock for various cross-coupling reactions in the synthesis of C-5mono-substituted thieno[2,3-d]pyrimidin-4-amines (e.g., 1b, 1h,1i, 1j, Table 1). It is noteworthy that fragment 20b was obtainedfrom 13 in four steps, with an average of 50–60% overall isolatedyield, using our protocol (Scheme 2). This methodology is signifi-cantly more efficient in preparing analogs of 1 that are selectivelymono-substituted at C-5 as compared to those reported previously(e.g., examples shown in Scheme 1).

In the course of these studies, we noted that thiophene 15 wassomewhat chemically unstable. Interestingly, protection of the2-amino moiety with N,N-dimethylformamide-dimethylacetal,provided intermediate 16, which is chemically stable and easy toprepare in high yield and purity (Scheme 2). Bromination of theprotected thiophene 16 was also carried-out in >80% yield, provid-ing intermediate 17 (Scheme 2). Compound 17 is a suitable build-ing block for a variety of cross-coupling reactions. We exploredselective cross-coupling with various boronic acids or boronateesters to obtain intermediates of general structure 18, which uponipso-iododesilylation at C-4 (i.e., leading to 19a), followed bySuzuki, Buchwald–Hartwig, Sonogashira, Stille or other cross-cou-pling reactions, gave the disubstituted thiophenes 19b.

Finally, cyclization of 19b provided a library of novel andstructurally diverse C-5/C-6 di-substituted thieno[2,3-d]pyrimi-

Figure 2. Inhibition data for human FPPS and GGPPS of select thienopyrimide-based bisphosphonates 2a–2f (R2, R5 and R6 substituents are selected from those shown inTable 1). (a) Superposition of analog 2e with the bioactive conformation of inhibitor 3 (PDB: 4DEM). The carbon skeleton of 2e and 3 are highlighted in light and dark greencolour, respectively; nitrogen, oxygen, sulphur and phosphorus atoms are indicated in blue, red, yellow and orange colour, respectively. (b) Structures of key thienopyrimide-based bisphosphonate compounds. (c) Inhibition data for hFPPS and hGGPPS at 10 lM of inhibitor; the hFPPS and hGGPPS inhibitors 3 and 4, respectively (Fig. 1) were used as+ve/�ve controls (% inhibition at 10 lM concentration of inhibitor; average values of three determinations; standard deviation ±10%).

2232 C.-Y. Leung et al. / Bioorg. Med. Chem. 21 (2013) 2229–2240

din-4-amines (1k–o, Table 1) in good overall yield. For example,preparation of analogs 1l from 15 was achieved in seven steps(including cross coupling under typical Suzuki and Sonogashiraconditions at C-5 and C-4, respectively) in an overall isolated yieldof 50%. Similarly, analog 1m (involving Buchwald–Hartwig amina-tion in the conversion of 19a–19b) was prepared from 15 in 30%isolated overall yield. It should be noted that these reactions werenot individually optimized during preparation of our library. Ana-log 1a (Table 1) was prepared by a different synthetic scheme,involving direct condensation of 2-aminothiophene-3-carbonitrilewith benzonitrile under basic condition and at high temperature,as previously reported.19

To gain some insight as to the potential for developing favour-able structure–activity-relationship (SAR) for thienopyrimidine-based inhibitors of hFPPS and/or hGGPPS, a small cluster of analogsfrom this library were converted to the corresponding bisphospho-nates 2 (Scheme 2). Treatment of fragments 1 with triethyl ortho-formate and diethylphosphite, followed by hydrolysis of thetetraethyl bisphosphonate esters with TMSBr/MeOH resulted inthe formation of bisphosphonic acids 2 (Scheme 2); details of thistwo-step protocol were previously described in the synthesis of thehFPPS inhibitor 3.21

2.2. Biological results and discussion

Initially, the bisphosphonic acid analogs 2a–f (Fig. 2b) wereevaluated in our routine hFPPS and hGGPPS inhibition assays at

a single concentration of 10 lM (as previously reported),21 alongwith inhibitors 3 (IC50 = 28 nM in hFPPS; IC50 >100 lM in hGGPPS)and 4 (IC50 = 410 nM in our own hGGPPS assay;21 IC50 >10 lM inhFPPS40) as the positive controls. The inhibition data observedare summarized in Figure 2c (% inhibition of hFPPS and hGGPPSat 10 lM of compound; average values of three determinations;standard deviation of approximately 10%). Significant inhibitionof hFPPS was observed with the parent molecule 2a, as well asthe C-2 and C-6 phenyl analogs 2b and 2d, respectively (75–80%inhibition at 10 lM). However, analog 2c exhibited <20% inhibi-tion at 10 lM (Fig. 2b and c). These preliminary data suggest thatC-2 and C-6 derivatives of the thienopyrimidine core may be ben-eficial in developing inhibitors of hFPPS, whereas favourable sub-stitution at C-5 may be limited. In order to gain further insightas to the volume of space within the hFPPS binding pocket occu-pied by the substituents at C-6, the tolyl and naphthyl moieties(2e and 2f, respectively) were also investigated. These modifica-tions resulted in modest improvement in inhibiting hFPPS(Fig. 2c). The full dose response for hFPPS inhibition was subse-quently determined for analogs 2e and 2f and their IC50 valueswere calculated to be 390 and 250 nM, respectively. Interestingly,this modest SAR optimization (i.e., compounds 2e and 2f) alsoimproved the selectivity in inhibiting hFPPS as compared tohGGPPS (Fig. 2c).

Superposition of the hFPPS-bound conformation of inhibitor 3(IC50 = 28 nM; PDB: 4DEM),21 with the thienopyrimidine analog2e strongly suggests that these compounds cannot adopt the same

C.-Y. Leung et al. / Bioorg. Med. Chem. 21 (2013) 2229–2240 2233

binding mode in the active site of hFPPS (Fig. 2a). Therefore, thethienopyrimidine-based bisphosphonates 2e and 2f representnovel leads for drug discovery into hFPPS inhibitors. To fully ex-plore the SAR potential of these compounds, synthesis of a largerand structurally diverse library of bisphosphonate analogs is cur-rently in progress. Concurrently, we are pursuing a number ofstructural investigations in order to determine the mechanism bywhich these compounds inhibit hFPPS.

3. Conclusion

Currently, nitrogen-containing bisphosphonates (N-BPs) are theonly clinically validated drugs that target the human FPPS, thusblocking prenylation.33,34 Although these drugs are mainly usedfor the treatment of bone-related diseases, recent clinical data pro-vides evidence that N-BPs are also disease modifying agents thatimprove the survival of cancer patients (particularly of patientswith multiple myeloma35) via mechanisms unrelated to their ske-letal effects. In an effort to identify compounds with improved bio-pharmaceutical properties (as compared to those of the currentdrugs), we designed a new class of bisphosphonates based on thethienopyrimidine scaffold 1. Thienopyrimidines are usually pre-pared via the Gewald method. However, this approach is not easilyamenable to parallel synthesis of highly substituted and structu-rally diverse analogs. In addition, structural diversity is typicallydictated by the starting ketones 5 (Scheme 1); thus the synthesisis linear and does not allow preparation of permutation libraries.Furthermore, selective mono-substitution at the Cb of the thio-phene sulfur is challenging due to the inherent higher reactivityof the Ca carbon. We modified the classical Gewald methodologyby employing 2-(1-(trimethylsilyl)-ethylidene)-malononitrile (14)as a novel synthon in the preparation of thieno[2,3-d]pyrimid-4-amine libraries (1; Scheme 2). Preparation of 5-iodothieno[2,3-d]pyrimidin-4-amine (20b) was achieved in high yields and signif-icantly fewer steps as compared to previous reports. This metho-dology was then used to prepare a small cluster ofthienopyrimidine-based bisphosphonate compounds (2) and probethe ability to such N-BPs to inhibit the hFPPS. In depth SAR studiesand structural investigations are currently in progress in order toevaluate the potential of these compounds for further optimizationinto potent and selective inhibitors of hFPPS.

4. Experimental

4.1. General procedures for characterization of compounds

All intermediate compounds were purified by normal phaseflash column chromatography on silica gel using a CombiFlash in-strument and the solvent gradient indicated. Key thienopyrimidinebuilding blocks, bisphosphonate esters and all final bisphosphonicacid inhibitors were analyzed by reverse-phase HPLC and fullycharacterized by 1H, 13C and 31P NMR, and MS (HR-MS for key frag-ments and final inhibitors). Chemical shifts (d) are reported in ppmrelative to the internal deuterated solvent (1H, 13C) or externalH3PO4 (d 0.00 31P), unless indicated otherwise. High-resolutionMS spectra were recorded using electrospray ionization (ESI+/�)and Fourier transform ion cyclotron resonance mass analyzer(FTMS). Melting points (when relevant) were determined using aconventional capillary melting point analyzer. The homogeneityof the bisphosphonate tetra esters and the final bisphosphonic acidinhibitors was confirmed by HPLC to >90% (using the conditions in-dicated below); IC50 values were determined only for samples with>95% homogeneity. HPLC analysis was performed using a WatersALLIANCE� instrument (e2695 with 2489 UV detector and 3100mass spectrometer).

Method (homogeneity analysis using a Waters Atlantis T3 C185 lm column):

Solvent A: H2O, 0.1% formic acid.Solvent B: CH3CN, 0.1% formic acid.Mobile phase: linear gradient from 95% A and 5% B to 5% A and95% B in 13 min, then 2 min at 100% B.Flow rate: 1 mL/min.

4.2. Synthesis of key fragments

4.2.1. 2-(1-(Trimethylsilyl)ethyl)malononitrile (14)Acetyltrimethylsilane (1.460 g, 12.56 mmol), malononitrile

(1.140 g, 12.56 mmol) and ammonium acetate (262.1 mg,2.386 mmol) were dissolved in acetic acid (0.58 mL, 10.05 mmol)and benzene (30 mL) in a 100 mL round bottom flask attached toa Dean–Stark trap and filled with benzene. The reaction mixturewas stirred and heated to 95 �C for 24 h. The resulting orange solu-tion was cooled and diluted with ethyl acetate (20 mL). The organiclayer was washed with saturated sodium bicarbonate solution(15 mL), water (45 mL), brine (15 mL) and dried over MgSO4. Theproduct was purified by column chromatography (25% ethyl acet-ate/hexanes) to give the desired product as clear pale yellow oil in90% yield (1.973 g).

1H NMR (400 MHz, CDCl3) d 2.34 (s, 3H), 0.36 (s, 9H).13C NMR (126 MHz, CDCl3) d 188.3, 113.2, 111.5, 94.4, 24.3,�2.2.HRMS (ESI�) Calcd for C8H11N2Si m/z [M�H]�: 163.06970,found m/z 163.06875 and HRMS (ESI�) Calcd 327.14667 forC16H23N4Si2, found m/z 327.14695 [2M�H]�.

4.2.2. 2-Amino-4-(trimethylsilyl)thiophene-3-carbonitrile (15)2-(1-(Trimethylsilyl)ethyl)malononitrile (1.21 g, 7.365 mmol)

and sulfur (248.02 mg, 7.734 mmol) were dissolved in pyridine(25 mL) at room temperature. To this, diethylamine (0.762 mL,7.365 mmol) was added dropwise. The reaction mixture stirredat room temperature for 18 h. Evaporation of pyridine affordedthe crude thiophene, which was dissolved in ethyl acetate(20 mL) and washed with water (45 mL), brine (15 mL), and driedover MgSO4. Purification by column chromatography (25% ethylacetate/hexanes, Rf = 0.58) afforded the desired product as an or-ange oil in 85% yield (803.5 mg).

1H NMR (300 MHz, CDCl3) d 6.38 (s, 1H), 4.74 (br s, 2H), 0.30 (s,9H).13C NMR (126 MHz, CDCl3) d 164.3, 141.1, 116.8, 116.7, 92.3,�1.3.HRMS (ESI+) Calcd 197.05632 for C8H13N2SSi, found m/z197.05615 [M+H]+.

4.2.3. 5-(Trimethylsilyl)thieno[2,3-d]pyrimidin-4-amine (20a)Fragment 20a was obtained in two different ways: (a) 2-Amino-

4-(trimethylsilyl)thiophene-3-carbonitrile (15a, 400 mg,2.04 mmol, 1 equiv) was added to formamide (8.1 mL, 200 mmol,100 equiv) in a pressure vessel. The reaction mixture was sealedand stirred at 145 �C in the dark for 16 h. (b) 3-Cyano-4-(trimethyl-silyl)thiophen-2-yl)-N,N-dimethylformimidamide (79.3 mg,0.315 mmol) was reacted with formamide (anhydrous, 2.5 mL,63.08 mmol) in a dry 15 mL pressure vessel. The vessel was flushedwith argon and the mixture stirred at 130 �C for 45 h. The dark redsolution was diluted with ethyl acetate, washed with water(25 mL), brine (10 mL), and dried over Na2SO4. The crude mixture

2234 C.-Y. Leung et al. / Bioorg. Med. Chem. 21 (2013) 2229–2240

was purified by flash column chromatography (5–30% EtOAc/hex-anes, dry loading) to afford the desired product as a pink solid in80% yield (59.0 mg).

1H NMR (400 MHz, CDCl3) d 8.47 (s, 1H), 7.43 (s, 1H), 5.36 (br s,2H), 0.46 (s, 9H).13C NMR (126 MHz, CDCl3) d 170.9, 158.8, 153.6, 133.2, 131.3,119.8, 0.4.HRMS (ESI+) Calcd 224.06722 for C8H13N2SSi, found m/z224.06705 [M+H]+.

4.2.4. 5-Iodothieno[2,3-d]pyrimidin-4-amine (20b)A solution of 5-(trimethylsilyl)thieno[2,3-d]pyrimidin-4-amine

(220.4 mg, 0.987 mmol, 1 equiv) in dichloromethane (2 mL) wasstirred at �10 �C ice slurry for 5 min. 1 M Iodine monochloride indichloromethane (2.96 mL, 2.96 mmol, 3 equiv) was added to thereaction mixture drop-wise and the reaction mixture was stirredat �10 �C for 30 min. Ice cooled water (30 mL) was directly addedto the reaction mixture to quench the reaction. Dichloromethane(2 � 20 mL) at room temperature was added to the mixture andthe entire mixture was filtered through a Whatman #5 2.5 lm fil-ter paper. The yellowish solid was washed with dichloromethane(2 � 10 mL) and recrystallized with methanol to give the desiredproduct 5-iodothieno[2,3-d]pyrimidin-4-amine as a yellow coloredsolid (271.9 mg, >98% yield).

1H NMR (300 MHz, CD3CN): d 8.33 (1H, s), 7.79 (1H, s), 7.44 (brs, 2H).13C NMR (126 MHz, acetone-d6) d 159.5, 154.7, 128.6, 116.4,106.1, 70.5.HRMS (ESI+) Calcd 277.92434 for C6H4IN3S, found m/z277.92353 [M+H]+.Melting point: 196 �C (dec).

4.2.5. 3-Cyano-4-(trimethylsilyl)thiophen-2-yl)-N,N-dimethylformimidamide (16)

To a solution of 2-amino-4-(trimethylsilyl)thiophene-3-carbo-nitrile (15, 372.40 mg, 1.90 mmol) in DMF (20 mL) was addedDMF–DMA (2.5 mL, 18.97 mmol). After stirring at room tempera-ture for 4 h, the reaction mixture was diluted with ethyl acetate,washed with water (60 mL), brine (20 mL), and dried over MgSO4.Solvent was removed in vacuo to afford the desired product as abrown-yellow solid in 90% yield (440.1 mg).

1H NMR (500 MHz, CDCl3) d 7.72 (s, 1H), 6.63 (s, 1H), 3.10 (s,3H), 3.09 (s, 3H), 0.32 (s, 9H).13C NMR (126 MHz, CDCl3) d 168.9, 154.8, 141.6, 120.7, 117.5,101.2, 40.7, 35.2, �1.3.29Si NMR (99 MHz, CDCl3) d �6.69.HRMS (ESI+) Calcd 252.09852 for C11H18N3SSi, found m/z252.09781 [M+H]+.

4.2.6. 5-Bromo-3-cyano-4-(trimethylsilyl)thiophen-2-yl-N,N-dimethylformimidamide (17)

N-Bromosuccinimide (121.2 mg, 0.68 mmol) was added to a so-lution of 3-cyano-4-(trimethylsilyl)thiophen-2-yl-N,N-dimethyl-formimidamide (163.0 mg, 0.648 mmol) in DMF (7 mL). Theyellow solution was stirred in the absence of light at room tem-perature for 13 h. The mixture was diluted with ethyl acetate,washed with water (25 mL), brine (10 mL), and dried over MgSO4.Solvent was removed in vacuo to afford the desired product as abrown-orange solid in 80% yield (177.1 mg).

1H NMR (500 MHz, CDCl3) d 7.63 (s, 1H), 3.09 (s, 6H), 0.44 (s,9H).13C NMR (126 MHz, CDCl3) d 168.4, 154.6, 138.9, 116.7, 106.2,101.9, 40.8, 35.3, 0.2.HRMS (ESI+) Calcd 330.00903 for C11H17N3BrSSi, found m/z330.00926 [M+H]+.

4.2.7. 5-Bromo-3-cyanothiophen-2-yl-N,N-dimethylformimidamide

A 1 M solution of TBAF (5.13 mL) in THF was added dropwise toa solution of 5-bromo-3-cyano-4-(trimethylsilyl)thiophen-2-yl-N,N-dimethylformimidamide (17, 1.61 g, 4.89 mmol) in THF(100 mL) cooled to 0 �C. The reaction mixture was warmed to roomtemperature and stirred in the dark for 3 h. The volume of THF wasreduced in vacuo and EtOAc was added, washed with water(3 � 50 mL), brine (25 mL), and dried over Na2SO4. The desiredcrude product was obtained as a red oil in 95% yield (1.20 g) andused as such for the synthesis of analogs 1, where R5 = H withoutany further purification.

1H NMR (400 MHz, CDCl3) d 7.64 (s, 1H), 6.86 (s, 1H), 3.11 (s,6H).13C NMR (300 MHz, CDCl3) d 167.0, 154.2, 128.3, 115.1, 99.4,96.5, 40.8, 35.2.MS (ESI+) m/z: 258.06 [M+H]+.

4.2.8. N0-(3-Cyano-4-iodo-5-phenylthiophen-2-yl)-N,N-dimethylformimidamide (19a, R5 = a)

Silver trifluoroacetate (93.2 mg, 0.42 mmol) was added to a so-lution of N0-(3-cyano-5-phenyl-4-(trimethylsilyl)thiophen-2-yl)-N,N-dimethylformimidamide (69.1 mg, 0.21 mmol) in THF(20 mL) cooled to �78 �C and stirred under argon for 15 min. Io-dine (214.2 mg, 0.84 mmol) dissolved in THF (10 mL) was addeddropwise to the cold mixture and stirred in the dark at �78 �Cfor 4 h. Ethyl acetate was added and the mixture was filteredthrough Celite. The filtrate was washed with 2 M sodium thiosul-fate, brine, and dried over Na2SO4. The crude mixture was purifiedby flash column chromatography (5–30% EtOAc/hexanes, solidloading) to afford the desired product as an orange solid in >98%yield (80.0 mg).

1H NMR (400 MHz, CDCl3) d 7.77 (s, 1H), 7.58–7.51 (m, 2H), 7.40(m, 3H), 3.13 (s, 6H).13C NMR (75 MHz, CDCl3) d 167.2, 154.7, 134.1, 130.7, 129.5,128.7, 128.6, 116.8, 105.7, 78.1, 40.9, 35.3.HRMS (ESI+) Calcd 381.98694 for C14H13N3IS, found m/z381.98667 [M+H]+.

4.3. Synthesis of thieno[2,3-d]pyrimidin-4-amines—generalprotocols

4.3.1. Suzuki coupling reactions using fragment 20bFragment 20b is not very soluble in non-polar solvents; conse-

quently all reactions were carried out in MeOH. To an argonflushed microwave reactor vial, fragment 20b, the boronic acid orboronate ester (1.4 equiv) and Pd(PPh3)4 (0.1 equiv) and KF(2.5 equiv) were added and flushed again with argon. Argonflushed methanol was added to the reaction mixture and the reac-tion was stirred at 120 �C for 20 min (120 W). The crude mixturewas filtered through celite and concentrated to dryness under va-cuum. The crude product was purified by normal phase flash col-umn chromatography on silica gel (silica gel was pre-washed

C.-Y. Leung et al. / Bioorg. Med. Chem. 21 (2013) 2229–2240 2235

with hexanes) using a CombiFlash instrument and a solvent gradi-ent from 100% hexanes to 100% EtOAc.

4.3.2. General protocol for the Suzuki coupling reactions usingfragment 17

In order to probe the versatility and stability of fragment 17, Suzu-ki coupling reactions were carried out using two different reactionconditions: (a) The boronic acid or boronate ester (1.5 equiv),Pd(PPh3)4 (0.1 equiv) and fragment 17 were dissolved in toluene/ethanol (3:1) (approximate concentration with respect to 17 of0.1 M). The mixture was degassed and flushed with Argon. Aqueous2 M Na2CO3 (2.5 equiv) was added and the mixture was again de-gassed and flushed with Argon. The reaction mixture was stirred at85 �C overnight. The crude was filtered through a plug of celite, rinsedwith 10 mL of solvent and concentrated under vacuum. The residuewas purified on silica gel using a CombiFlash instrument to give thedesired products (the common solvent gradient was from 2% EtOAcin hexanes to 100% EtOAc, unless otherwise indicated). (b) The proto-col described for the Suzuki reactions using fragment 20b was alsoused successfully; presumably the TMS group survives these condi-tions due to high solvation of the fluoride ion in methanol.

4.3.3. General protocol for Stille cross-coupling reactions usingfragment 19a

Palladium acetate (1 equiv) and XPhos (2 equiv) in DME (2 mL)were heated under argon to 85 �C in a 2-dram vial for 10 min. Tothis mixture cesium carbonate (2 equiv), and fragment 19a(1 equiv 0.3 mmol) were added and stannane (1.77 equiv). The vialwas flushed with argon and the reaction mixture was stirred at80 �C for 15 h. The reaction mixture was diluted with EtOAc(10 mL), washed with water (3 � 10 mL) and brine (10 mL), driedover Na2SO4, and concentrated under vacuum. The crude residuewas purified by column chromatography on silica gel using a Com-biFlash instrument and a solvent gradient from% EtOAc in hexanesto 100% EtOAc, unless otherwise indicated.

4.3.4. General protocol for Sonogashira cross-coupling reactionsusing fragment 19a

Triethylamine (0.5 mL) was added to a vial of the iodide 19a(0.081 mmol), acetylene (0.018 mL, 0.16 mmol), copper(II) bro-mide (2.7 mg, 0.012 mmol), and Pd(PPh3)4 (9.4 mg, 0.008 mmol).The vial was purged with argon, capped, and heated to 90 �C for13 h. Extracted with EtOAc (3 � 10 mL) and washed with saturatedsodium bicarbonate (5 mL), water (3 � 5 mL), brine (5 mL), anddried over Na2SO4. The crude mixture was purified by flash columnchromatography on silica gel (solid loading) using a solvent gradi-ent from 2% EtOAc in hexanes to 100% EtOAc (unless otherwise in-dicated) to afford the desired product.

4.3.5. General protocol for Buchwald–Hartwig aminationreactions using fragment 19a

The amine (5 equiv) was added to a degassed solution of frag-ment 19a (usually on a 0.03 mmol scale), Pd2(dba)3 (5 mol %),XantPhos (11 mol %), and cesium carbonate (1.7 equiv) in toluene(1 mL). The vial was purged with argon and the reaction mixturestirred at 100 �C for 18 h. A second portion of Pd2(dba)3 (5 mol %)and XantPhos (11 mol %) were added and the reaction mixturewas stirred at 100 �C for an additional 18 h. The reaction mixturewas diluted with EtOAc (10 mL), washed with water (3 � 10 mL)and brine (10 mL), dried over Na2SO4, and concentrated under va-cuum. The crude residue was purified by column chromatographyon silica gel using a CombiFlash instrument and a solvent gradient

from 2% EtOAc in hexanes to 100% EtOAc (unless otherwise indi-cated) to afford the desired product.

4.3.6. General protocol for the cyclization of C-4 and/or C-5substituted fragments 18 or 19b to thieno[2,3-d]pyrimidin-4-amines 1

The mono-substituted or di-substituted fragments 18 or 19b,respectively, (0.04 mmol) and dry formamide (excess, >200 equiv)were added to a dry 15 mL pressure vessel. The vessel was flushedwith argon and the mixture stirred at 130 �C for 48 h. The dark redsolution was diluted with EtOAc, washed with water (25 mL), brine(10 mL), and dried over Na2SO4. The crude mixture was purified byflash column chromatography (5–100% EtOAc/hexanes, solid load-ing) to afford the desired product.

4.4. Spectral data of key synthetic intermediates 18

The R5 groups indicated are taken from the fragments shown inTable 1.

4.4.1. N0-(3-Cyano-5-phenyl-4-(trimethylsilyl)thiophen-2-yl)-N,N-dimethylformimidamide (18, R5 = a)

Isolated as a beige solid in 90% yield (179 mg).

1H NMR (400 MHz, CDCl3) d 7.71 (s, 1H), 7.36–7.33 (m, 5H), 3.12(s, 3H), 3.08 (s, 3H), 0.12 (s, 9H).13C NMR (126 MHz, CDCl3) d 167.8, 154.6, 139.1, 136.3, 135.9,130.6, 128.5, 128.1, 118.0, 102.7, 40.7, 35.2, 0.4.HRMS (ESI+) Calcd 328.12982 for C17H22N3SSi, found m/z328.12920 [M+H]+.

4.4.2. N0-(3-Cyano-5-(p-tolyl)-4-(trimethylsilyl)thiophen-2-yl)-N,N-dimethylformimidamide (18, R5 = c)

Isolated as a pale yellow solid in 75% yield (115 mg).

1H NMR (400 MHz, CDCl3) d 7.79 (s, 1H), 7.21 (d, J = 8 Hz, 2H),7.15 (d, J = 8 Hz, 2H), 3.32 (s, 3H), 3.20 (s, 3H), 2.38 (s, 3H),0.14 (s, 9H).13C NMR (126 MHz, CDCl3) d 167.6, 154.5, 139.3, 138.3, 135.9,132.8, 130.4, 128.7, 117.9, 102.6, 40.6, 35.0, 21.4, 0.5.HRMS (ESI+) Calcd 342.14547 for C18H24N3SSi, found m/z342.14458 [M+H]+.

4.4.3. N0-(3-Cyano-5-(4-(trifluoromethyl)phenyl)-4-(trimethylsilyl)thiophen-2-yl)-N,N-dimethylformimidamide(18, R5 = e)

Isolated as a pale brown solid in 71% yield (49.6 mg).

1H NMR (300 MHz, CDCl3) d 7.72 (s, 1H), 7.61 (d, J = 8 Hz, 2H),7.46 (d, J = 8 Hz, 2H), 3.12 (d, J = 12 Hz, 6H), 0.14 (s, 9H).13C NMR (75 MHz, CDCl3) d 168.3, 154.7, 139.7, 137.4, 136.8,130.5 (q, JCF = 33 Hz), 125.0 (q, JCF = 4 Hz), 124.1 (q,JCF = 272 Hz), 117.7, 103.1, 40.8, 35.2, 0.5.HRMS (ESI+) Calcd 396.11775 for C18H21F3N3SSi, found m/z396.11519 [M+H]+.

4.4.4. N0-(3-Cyano-5-(naphthalen-2-yl)-4-(trimethylsilyl)thiophen-2-yl)-N,N-dimethylformimidamide(18, R5 = f)

Isolated as an orange solid in 63% yield (72.1 mg).

2236 C.-Y. Leung et al. / Bioorg. Med. Chem. 21 (2013) 2229–2240

1H NMR (400 MHz, CDCl3): d 7.87–7.81 (m, 5H), 7.74 (s, 1H),7.53–7.51 (m, 2H), 7.46 (m, 1H), 3.15 (s, 3H), 3.10 (s, 3H) 0.13(9H, s).13C NMR (75 MHz, CDCl3): d 167.9, 154.6, 139.1, 136.6, 133.3,133.0, 132.8, 129.6, 128.4, 128.1, 127.9, 127.7, 126.7, 126.6,40.7, 35.2, 0.6.HRMS (ESI+) Calcd 378.14547 for C21H24N3SSi, found m/z378.14412 [M+H]+.

4.4.5. N0-(4-Cyano-3-(trimethylsilyl)-[2,30-bithiophen]-5-yl)-N,N-dimethylformimidamide(18, R5 = i)

Isolated as light brown solid in 75% yield (53.1 mg).

1H NMR (400 MHz, CDCl3): d 7.70 (s, 1H), 7.31–7.29 (m, 1H),7.23 (m, 1H), 3.12 (3H, s), 3.09 (3H, s), 0.15 (s, 9H).13C NMR (75 MHz, CDCl3): d 167.6, 154.7, 154.6, 137.3, 135.7,132.9, 130.0, 125.5, 125.3, 117.8, 102.5, 40.7, 35.2, 0.2.HRMS (ESI+) Calcd 334.08624 for C15H20N3S2Si, found m/z334.08530 [M+H]+.

4.4.6. N0-(3-Cyano-4-(trimethylsilyl)-5-vinylthiophen-2-yl)-N,N-dimethylformimidamide (18, R5 = h)

Isolated as beige solid in 75% yield (63 mg) and 95% purity (asdetermined by LC–MS). The impurity was determined to be the de-halogenated thiophene, however, this compound was eliminatedfrom the library for the subsequent steps.

1H NMR (400 MHz, CDCl3) d 7.72 (s, 1H), 6.86 (dd, J = 16.9,10.8 Hz, 1H), 5.36 (d, J = 17.0 Hz, 1H), 5.10 (d, J = 10.8 Hz, 1H),3.12 (s, 3H), 3.10 (s, 3H), 0.41 (s, 9H).13C NMR (75 MHz, CDCl3) d 166.3, 154.8, 138.1, 137.5, 130.7,117.5, 113.6, 103.0, 40.7, 35.1, 0.8.HRMS (ESI+) Calcd 278.11417 for C13H20N3SSi, found m/z278.11400 [M+H]+.

4.5. Spectral data of key synthetic intermediates 19

The R4 and/or R5 groups indicated are taken from the fragmentsshown in Table 1.

4.5.1. N0-(3-Cyano-5-phenyl-4-(pyrazin-2-yl)thiophen-2-yl)-N,N-dimethylformimidamide (19b, R4 = b, R5 = a)

Isolated as beige solid in 41% yield (8.0 mg) and 95% purity (asdetermined by LC–MS).

1H NMR (400 MHz, CDCl3): d 8.67 (dd, J = 2.6, 1.6 Hz, 1H), 8.46(d, J = 2.6 Hz, 1H), 8.36 (d, J = 1.5 Hz, 1H), 7.85 (s, 1H), 7.28–7.25 (m, 3H), 7.17–7.14 (m, 2H), 3.15 (s, 3H), 3.15 (s, 3H).13C NMR (75 MHz, CDCl3): d 166.9, 154.6, 149.3, 145.8, 144.5,143.1, 132.7, 132.2, 131.1, 129.3, 129.0, 128.4, 115.9, 99.2,40.9, 35.3.HRMS (ESI+) Calcd 334.11209 for C18H16N5S, found m/z334.11144 [M+H]+.

4.5.2. N0-(3-Cyano-5-phenyl-4-(phenylethynyl)thiophen-2-yl)-N,N-dimethylformimidamide (19b, R4 = g, R5 = a)

Isolated as a red-orange solid in 95% yield (28.0 mg).

1H NMR (400 MHz, CDCl3) d 7.86–7.75 (d, J = 7.4 Hz, 3H), 7.83 (s,1H), 7.57–7.51 (m, 2H), 7.42 (t, J = 7.5 Hz, 2H), 7.37–7.30 (m,4H), 3.15 (s, 3H), 3.15 (s, 3H).

13C NMR (126 MHz, CDCl3) d 164.2, 154.6, 134.0, 133.3, 131.9,128.8, 128.7, 128.5, 128.2, 127.5, 122.9, 116.6, 115.5, 100.9,94.7, 83.3, 40.9, 35.3.HRMS (ESI+) Calcd 356.12159 for C22H18N3S, found m/z356.12098 [M+H]+.

4.5.3. N0-(3-Cyano-4-(cyclopropylethynyl)-5-phenylthiophen-2-yl)-N,N-dimethylformimidamide (19b, R4 = h, R5 = a)

Isolated beige solid in 90% yield (15 mg, based on the recoveryof some starting material).

1H NMR (500 MHz, CDCl3) d 7.78–7.75 (m, 3H), 7.38–7.35 (m,2H), 7.30–7.26 (m, 1H), 3.10 (s, 6H), 1.52–1.46 (m, 1H), 0.91–0.87 (m, 2H), 0.87–0.83 (m, 2H).13C NMR (126 MHz, CDCl3) d 163.8, 154.5, 133.30, 132.9, 128.6,127.9, 127.1, 117.2, 115.6, 101.4, 99.6, 69.4, 40.9, 35.2, 9.1, 0.7.HRMS (ESI+) Calcd 320.12159 for C19H18N3S, found m/z320.12139 [M+H]+.

4.5.4. N0-(3-Cyano-4-morpholino-5-phenylthiophen-2-yl)-N,N-dimethylformimidamide (19b, R4 = j, R5 = a)

Isolated as a beige solid in 90% yield (9.5 mg).

1H NMR (400 MHz, CDCl3) d 7.74 (s, 1H), 7.50 (d, J = 7.3 Hz, 2H),7.35 (t, J = 7.5 Hz, 2H), 7.29 (t, J = 7.3 Hz, 1H), 3.75–3.71 (m, 4H),3.12 (s, 3H), 3.10 (s, 3H), 3.10–3.07 (m, 4H).13C NMR (126 MHz, CDCl3) d 164.4, 153.9, 143.8, 133.6, 129.3,128.4, 127.6, 118.4, 116.3, 96.6, 67.5, 51.7, 40.9, 35.2.HRMS (ESI+) Calcd 341.14306 for C18H21ON4S, found m/z341.14218 [M+H]+.

4.5.5. N0-(4-(Benzyl(methyl)amino)-3-cyano-5-phenylthiophen-2-yl)-N,N-dimethylformimidamide (19b, R4 = k, R5 = a)

The crude residue was purified by column chromatography on aCombiFlash instrument using a solvent gradient from 3% EtOAc/hexanes to 100% EtOAc with 0.1% triethylamine. The desired pro-duct was obtained in 45% yield (19 mg), 70% purity (as determinedby 1H NMR). The impurity was determined to be the dehaloge-nated thiophene and was eliminated in the following step.

1H NMR (400 MHz, CDCl3) d 7.76 (s, 1H), 7.50–7.44 (m, 1H),7.38–7.27 (m, 5H), 7.25–7.21 (m, 4H), 4.16 (s, 2H), 3.14–3.07(m, 8H), 2.74 (s, 3H).13C NMR (75 MHz, CDCl3) d 164.2, 153.9, 144.6, 138.4, 133.7,129.1, 128.9, 128.3, 128.2, 127.6, 127.3, 127.1, 125.3, 121.9,120.1, 116.6, 96.9, 60.1, 41.1, 35.2.HRMS (ESI+) Calcd 375.16379 for C22H23N4S, found m/z375.16296 [M+H]+.

4.6. Spectral data of key thieno[2,3-d]pyrimidin-4-amines (1)

The yields indicated are for the isolated pure fragments after thecyclization step of 19b–1 from the library synthesis. For the keybuilding blocks 5-(trimethylsilyl)thieno[2,3-d]pyrimidin-4-amine(20a) and 5-iodothieno[2,3-d]pyrimidin-4-amine (20b), refer toSections 4.2.3 and 4.2.4, respectively.

4.6.1. 2-Phenylthieno[2,3-d]pyrimidin-4-amine (1a)Synthesis of derivative 20c was achieved via direct condensa-

tion of 2-aminothiophene-3-carbonitrile with benzonitrile, aspreviously reported.12b To an argon flushed pressure vessel, 2-

C.-Y. Leung et al. / Bioorg. Med. Chem. 21 (2013) 2229–2240 2237

aminothiophene-3-carbonitrile (150 mg, 1.21 mmol, 1 equiv),benzonitrile (174.4 mg, 1.69 mmol, 1.4 equiv) and potassiumtert-butoxide (135.3 mg, 1.21 mmol, 1 equiv) were added andthe mixture was flushed again with argon. Anhydrous dioxane(1 mL) was added and the reaction mixture was stirred at150 �C for 14 h. The crude mixture was concentrated to drynessunder vacuum and was purified by flash column chromatogra-phy (0–50% EtOAc/hexanes, solid loading) to afford the final pro-duct in 27% yield (37.7 mg).

1H NMR (500 MHz, CDCl3): d 8.45–8.42 (m, 2H), 7.48–7.45 (m,3H), 7.26 (d, J = 6.0 Hz, 1H), 7.14 (d, J = 6.0 Hz, 1H), 5.55 (s, 2H).13C NMR (126 MHz, CDCl3): d 168.9, 160.3, 157.8, 137.9, 130.1,128.4, 128.2, 123.3, 117.4, 114.3.MS (ESI+) m/z: 228.1 [M+H]+.

4.6.2. 5-Phenylthieno[2,3-d]pyrimidin-4-amine (1b)Isolated as pale yellow solid in 22% yield (27 mg).

1H NMR (500 MHz, CD3OD): d 8.29 (s, 1H), 7.51–7.46 (m, 5H),7.31 (s, 1H).13C NMR (126 MHz, CD3OD) d 168.3, 160.0, 154.6, 137.2, 136.8,130.2, 130.1, 129.8, 122.1, 114.9.MS (ESI+) m/z: 228.4 [M+H]+.

4.6.3. 6-Bromothieno[2,3-d]pyrimidin-4-amine (1c)Isolated as a pale yellow solid in 35% yield (321 mg).

1H NMR (300 MHz, DMSO-d6): d 8.22 (1H, s), 7.72 (1H, s), 7.56(2H, s).13C NMR (300 MHz, DMSO-d6): d 167.5, 157.7, 154.8, 123.3,116.7, 109.7.MS (ESI+) m/z: 229.9 and 232.0 [M+H]+.

4.6.4. 6-(p-Tolyl)thieno[2,3-d]pyrimidin-4-amine (1d)Isolated as a beige solid in 65% yield (49.6 mg).

1H NMR (400 MHz, DMSO-d6): d 8.28 (s, 1H), 8.18 (s, 1H), 8.15(s, 1H), 8.06–8.01 (m, 2H), 7.96–7.94 (m, 1H), 7.84–7.83 (m,1H), 7.62–7.53 (m, 4H).13C NMR (75 MHz, DMSO-d6) d 165.5, 162.9, 158.2, 154.0, 138.2,130.5, 129.9, 125.5, 117.0, 114.9, 20.8.HRMS (ESI+) Calcd for C13H12N3S m/z [M+H]+: 242.07464, foundm/z 242.07383.

4.6.5. 6-(4-(Trifluoromethyl)phenyl)thieno[2,3-d]pyrimidin-4-amine (1e)

Isolated as a pale yellow solid in 56% yield (18.6 mg).

1H NMR (500 MHz, DMSO-d6): d 8.29 (s, 1H), 8.14 (s, 1H), 7.86(s, 4H), 7.66 (s, 2H). 13C NMR (126 MHz, DMSO-d6) d 166.3,158.5, 154.6, 137.1, 135.7, 128.31 (q, J = 32.0 Hz), 126.3 (q,J = 3.7 Hz), 124.09 (q, J = 272.0 Hz), 117.8, 116.9.HRMS (ESI+) Calcd 296.04638 for C13H9F3N3S, found m/z296.04552 [M+H]+.

4.6.6. 6-(Naphthalen-2-yl)thieno[2,3-d]pyrimidin-4-amine (1f)Isolated as a brown solid in 35% yield (15.4 mg).

1H NMR (500 MHz, DMSO-d6): d 8.28 (s, 1H), 8.18 (s, 1H), 8.15(s, 1H), 8.06–8.01 (m, 2H), 7.96–7.94 (m, 1H), 7.84–7.83 (m,1H), 7.62–7.53 (m, 4H). 13C NMR (126 MHz, DMSO-d6) d165.9, 158.3, 154.3, 133.0, 132.6, 130.7, 129.0, 128.1, 127.7,127.0, 126.7, 124.4, 123.4, 117.1, 116.3, 104.6.HRMS (ESI+) Calcd 278.07464 for C16H11N3S, found m/z278.07371 [M+H]+.

4.6.7. 6-(Thiophen-3-yl)thieno[2,3-d]pyrimidin-4-amine (1g)Isolated as a brown solid in 28% yield (9.6 mg).

1H NMR (400 MHz, DMSO-d6): d 8.24 (s, 1H), 7.78 (m, 2H), 7.73(dd, J = 5.0, 2.9 Hz, 1H), 7.50 (br s, 2H), 7.41 (dd, J = 5.0, 1.4 Hz, 1H).13C NMR (126 MHz, DMSO-d6) d 165.3, 158.1, 154.0, 134.6,133.1, 128.3, 125.4, 121.9, 116.6, 115.3.HRMS (ESI+) Calcd 234.01542 for C10H7N3S2, found m/z234.01433 [M+H]+.

4.6.8. 5-(4-Nitrophenyl)thieno[2,3-d]pyrimidin-4-amine (1h)Isolated as orange solid in 40% yield (14.2 mg); all NMR data

was consistent with previous reports.24

1H NMR (500 MHz, DMSO-d6): d 8.37 (s, 1H), 8.34 (d, J = 8.2 Hz,2H), 7.73 (d, J = 8.2 Hz, 2H), 7.69 (s, 1H), 7.06 (s, 2H).13C NMR (75 MHz, CDCl3) d 167.7, 158.3, 154.0, 147.1, 142.0,133.2, 130.1, 123.9, 122.9, 112.2.MS (ESI+) m/z: 273.1 [M+H]+.

4.6.9. 5-(4-(Trifluoromethyl)phenyl)thieno[2,3-d]pyrimidin-4-amine (1i)

Isolated as a pale yellow solid in 57% yield (28.2 mg).

1H NMR (400 MHz, DMSO-d6): d 8.36 (s, 1H), 7.87 (d, J = 8.0 Hz),7.70 (d, J = 8.0 Hz), 7.62 (s, 1H).13C NMR (126 MHz, DMSO-d6) d 167.5, 158.3, 153.9, 139.6,133.6, 129.1, 128.5 (q, J = 32.1 Hz), 125.7 (q, J = 3.8 Hz), 124.3(q, J = 272.1 Hz), 122.2, 112.4.HRMS (ESI+) Calcd 296.04638 for C13H8F3N3S, found m/z294.04552 [M+H]+.

4.6.10. 5-(Naphthalen-2-yl)thieno[2,3-d]pyrimidin-4-amine (1j)Isolated as a pale yellow solid in 54% yield (27.0 mg).

1H NMR (500 MHz, DMSO-d6): d 8.37 (s, 1H), 8.08–8.00 (m, 4H),7.62–7.59 (m, 4H).13C NMR (75 MHz, DMSO-d6) d 167.3, 158.4, 153.8, 134.9, 133.0,132.8, 132.4, 128.5, 128.0, 127.8, 127.7, 126.8, 126.7, 126.6,121.1, 113.0.HRMS (ESI+) Calcd 278.07464 for C16H11N3S, found m/z278.07371 [M+H]+.

4.6.11. 6-Phenyl-5-(phenylethynyl)thieno[2,3-d]pyrimidin-4-amine (1k)

Isolated as a brown solid in 85% yield (12 mg).

1H NMR (500 MHz, CDCl3) d 8.45 (s, 1H), 7.96–7.94 (m, 1H),7.94–7.93 (m, 1H), 7.51–7.47 (m, 4H), 7.45–7.37 (m, 4H),�6.30 (br s, 2H).

2238 C.-Y. Leung et al. / Bioorg. Med. Chem. 21 (2013) 2229–2240

13C NMR (126 MHz, CDCl3) d 165.3, 158.4, 154.6, 143.9, 133.0,131.4, 129.4, 129.3, 129.0, 128.9, 128.6, 122.0, 116.5, 108.8,94.8, 85.2.HRMS (ESI+) Calcd 328.09029 for C20H14N3S, found m/z328.09015 [M+H]+.

4.6.12. 5-(Cyclopropylethynyl)-6-phenylthieno[2,3-d]pyrimidin-4-amine (1l)

Isolated as a beige solid in 85% yield (11 mg).

1H NMR (500 MHz, CDCl3): d 8.39 (s, 1H), 7.88–7.81 (m, 2H),7.49–7.35 (m, 3H), 1.52 (m, 1H), 0.98–0.92 (m, 2H), 0.84 (m,2H).13C NMR (126 MHz, CDCl3): d 164.9, 158.4, 154.4, 143.0, 133.1,129.1, 128.8, 128.4, 116.7, 109.3, 99.5, 71.6, 8.8, 0.55.HRMS (ESI+) Calcd 292.09029 for C17H14N3S, found m/z292.09030 [M+H]+.

4.6.13. 5-Morpholino-6-phenylthieno[2,3-d]pyrimidin-4-amine(1m)

The crude mixture was purified by flash column chromatogra-phy (5–100% EtOAc/hexanes with 0.1% Et3N, solid loading) to af-ford the desired product as a beige solid in 50% yield (20.5 mg).

1H NMR (400 MHz, CDCl3) d 8.42 (s, 1H), 7.49–7.41 (m, 5H), 3.84(d, J = 10.6 Hz, 2H), 3.60 (td, J = 11.5, 2.3 Hz, 2H), 3.03 (td,J = 11.6, 2.8 Hz, 2H), 2.95 (d, J = 11.8 Hz, 2H), 1.60 (s, 1H).13C NMR (75 MHz, CDCl3) d 164.5, 158.8, 154.3, 138.3, 133.4,131.2, 131.0, 129.2, 128.6, 113.9, 67.8, 53.2.HRMS (ESI+) Calcd 313.11176 for C16H17ON4S, found m/z313.11125 [M+H]+.

4.6.14. N5-Benzyl-N5-methyl-6-phenylthieno[2,3-d]pyrimidine-4,5-diamine (1n)

The crude mixture was purified by flash column chromatogra-phy (5–100% EtOAc/hexanes with 0.1% Et3N, solid loading) to af-ford the desired product as a pale orange solid in 53% yield(9.0 mg).

1H NMR (500 MHz, CDCl3) d 8.41 (s, 1H), 7.47–7.36 (m, 5H),7.29–7.21 (m, 3H), 7.12–7.06 (m, 2H), 3.99 (d, J = 13.3 Hz, 1H),3.66 (d, J = 13.3 Hz, 1H), 2.77 (s, 3H).13C NMR (126 MHz, CDCl3) d 164.4, 158.6, 154.3, 139.3, 138.1,133.6, 130.8, 130.6, 128.9, 128.9, 128.7, 128.46, 127.7, 114.2,60.7, 43.4.HRMS (ESI+) Calcd 347.13249 for C20H19N4S, found m/z347.13188 [M+H]+.

4.6.15. 6-Phenyl-5-(pyrazin-2-yl)thieno[2,3-d]pyrimidin-4-amine (1o)

Isolated as a beige solid in 95% yield (7.0 mg).

1H NMR (500 MHz, CDCl3) d 8.68 (dd, J = 2.6, 1.5 Hz, 1H), 8.50 (d,J = 2.6 Hz, 1H), 8.48 (s, 1H), 8.29 (d, J = 1.4 Hz, 1H), 7.36–7.30(m, 3H), 7.22–7.19 (m, 2H).13C NMR (126 MHz, CDCl3) d 167.3, 159.1, 154.1, 150.8, 149.0,143.1, 143.0, 141.9, 132.9, 130.2, �129.4 (3 � C), 126.1, 115.9.HRMS: Calcd 306.08079 for C16H12N5S, found m/z 306.08165[M+H]+.

4.7. Synthesis of bisphosphonic acids 2

4.7.1. Standard 2-step procedure for the conversion ofthienopyrimidin-4-amines 1 to inhibitors 2

The R2, R5 and R6 groups indicated are taken from the fragmentsshown in Table 1.

Step (a): A solution of the thienopyrimidin-4-amine 1(0.2 mmol) in anhydrous toluene (10 mL) was flushed with argon.Triethyl orthoformate (1.5 equiv) and diethylphosphite (7 equiv)was added to the reaction mixture via syringe and the reactionmixture was flushed again with argon, sealed, and stirred for130 �C in the dark for 48 h. The crude mixture was concentratedto dryness under vacuum and purified by flash column chromato-graphy (20–100% EtOAc/hexanes to 0–20% MeOH/EtOAc, solidloading) to afford the desired tetraethyl bisphosphonate ester.

Step (b): A solution of the tetraethyl bisphosphonate ester(1 equiv) in CH2Cl2 was cooled to 0 �C and trimethylsilyl bromide(15 equiv) was added via syringe. The reaction mixture was stirredat room temperature for 3–5 days; completion of conversion wasmonitored by 31P NMR. The mixture was then concentrated undervacuum, diluted with HPLC grade MeOH (�5 mL), and the solventwas evaporated to dryness; this step was repeated four times. Theorganic solvents were evaporated under vacuum, the residue wassuspended in 0.5 mL MeOH, excess water (�5 mL Milli-Q grade)was added to induce full precipitation of the final bisphosphonicacid. The amorphous powder was collected by filtration, washedwith de-ionized water (2�), with HPLC-grade CH3CN (2�), withdistilled Et2O or toluene (2�) and dried under vacuum to givethe final compound as a white solid.

4.7.2. (Thieno[2,3-d]pyrimidin-4-ylamino)methylenebisphosphonic acid (2a)

The tetraethyl ((5-(trimethylsilyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene bisphosphonate was prepared from inter-mediate 20a; isolated in 60% yield (36.7 mg).

1H NMR (400 MHz, CD3OD): d 8.50 (s, 1H), 7.45 (s, 1H), 5.98 (td,J = 22.0, 9.9 Hz, 1H), 5.78 (d, J = 10.3 Hz, 1H), 4.28–4.12 (m, 8H),1.26 (td, J = 7.1, 1.7 Hz, 12H), 0.52 (s, 9H).13C NMR (126 MHz, CD3OD): d 170.1, 156.2, 152.7, 133.0, 131.3,120.3, 63.4 (d, JCP = 122.5 Hz), 44.2 (t, JCP = 583 Hz), 16.3, 0.09.31P NMR (81 MHz, CD3OD): d 14.81.MS (ESI) m/z 532.15 [M+Na]+.

A solution of the bisphosphonate tetraester (47.4 mg,0.093 mmol) in THF (2 mL) was then cooled to 0 �C and 0.1 mL(23.6 mg of a 1 M solution, 0.1 mmol) of TBAF in THF was added.After stirring for 20 min at 0 �C, the mixture was warmed to ambi-ent temperature and stirred for and additional 4 h in the dark. THFwas removed under vacuum and the crude mixture was dissolvedin 10 mL of EtOAc, washed with water (1 � 5 mL) and brine(1 � 5 mL), and dried over anhydrous sodium sulfate. The desily-lated product was obtained quantitatively as a pale yellow solid(41.2 mg).

1H NMR (500 MHz, CD3OD): d 8.47 (s, 1H), 7.72 (d, J = 6.0 Hz,1H), 7.57 (d, J = 6.0 Hz 1H), 6.00 (t, J = 23.7 Hz, 1H), 4.20 (m,8H), 1.26 (m, 12H).13C NMR (126 MHz, CD3OD): d 167.6, 157.6, 154.1, 125.1, 119.8,118.5, 65.16, 45.6 (t, JCP = 150.1 Hz), 16.6.31P NMR (81 MHz, CD3OD): d 18.52.HRMS (ESI+) Calcd 460.08370 for C15H25N3NaO6P2S, found461.08320 [M+Na]+.

C.-Y. Leung et al. / Bioorg. Med. Chem. 21 (2013) 2229–2240 2239

The final inhibitor, thieno[2,3-d]pyrimidin-4-ylamino methy-lene bisphosphonic acid (2a) was isolated as a white solid in 70%yield (20.8 mg).

1H NMR (400 MHz, D2O, 10% ND4OD, 10% DMSO internal stan-dard): d 8.284 (s, 1H), 7.549 (d, J = 5.9 Hz, 1H), 7.427 (d,J = 6.1 Hz, 1H), 4.594 (t, J = 19.1 Hz, 1H).13C NMR (126 MHz, D2O, 20% ND4OD, 10% DMSO internal stan-dard): d 164.3, 157.3, 154.2, 123.4, 119.8, 117.9, 51.7 (t,JCP = 125.6 Hz).31P NMR (81 MHz, D2O, 20% ND4OD): d 13.308.HRMS (ESI�) Calcd 323.96873 for C7H8SN3O6P2, found323.96125 [M�H]�.

4.7.3. (((2-Phenylthieno[2,3-d]pyrimidin-4-yl)amino)methylene)diphosphonic acid (2b)

The tetraethyl (((2-phenylthieno[2,3-d]pyrimidin-4-yl)amino)-methylene)bis(phosphonate) was isolated as an orange oil 61%(52.0 mg).

1H NMR (500 MHz, MeOD-d4): d 8.43–8.41 (m, 2H), 7.71 (d,J = 6.0 Hz, 1H), 7.51 (d, J = 6.0 Hz, 1H), 7.49–7.47 (m, 4H), 6.21(t, J = 23.7 Hz, 1H), 4.22–4.19 (m, 8H), 1.25–1.22 (m, 12H).13C NMR (126 MHz, MeOD-d4): d 169.5, 160.5, 157.3, 139.0,131.5, 129.5, 129.1, 124.8, 119.7, 116.8, 65.1 (m), 45.5 (t,J = 150.1 Hz), 16.7.31P NMR (81 MHz, MeOD-d4): d 17.6.MS (ESI+) m/z: 514.7 [M+H]+.

The final product (((2-phenylthieno[2,3-d]pyrimidin-4-yl)ami-no)methylene)diphosphonic acid (2b) was isolated as a pale yellowsolid in 66% yield (22.0 mg).

1H NMR (300 MHz, D2O): d 8.15–8.12 (m, 2H), 7.44–7.40 (m,4H), 7.28 (d, J = 6.0 Hz, 1H), 4.86 (t, J = 19.0 Hz, 1H).13C NMR (126 MHz, D2O): d 166.3, 161.5, 157.8, 138.4, 131.4,129.6, 129.1, 128.8, 123.6, 119.9, 116.6, 105.9, 50.6 (t, J = 123.6).31P NMR (81 MHz, D2O): d 14.2.HRMS (ESI�) Calcd 399.9928 for C13H12N3P2O6S, found m/z399.9922 [M�H]�.

4.7.4. (((5-Phenylthieno[2,3-d]pyrimidin-4-yl)amino)methylene)diphosphonic acid (2c)

The tetraethyl (((5-phenylthieno[2,3-d]pyrimidin-4-yl)amino)-methylene)bis(phosphonate) was isolated as a pale yellow solid24% yield (61 mg).

1H NMR (500 MHz, MeOD-d4): d 8.52 (s, 1H), 7.60–7.55 (m, 3H),7.52–7.50 (m, 2H), 7.45 (s, 1H), 5.80 (t, J = 22.4 Hz, 1H), 4.11–4.03 (m, 8H), 1.22–1.19 (m, 12H).13C NMR (75 MHz, MeOD-d4): d 166.9, 152.8, 135.4, 134.4,129.2, 128.9, 128.8, 122.0, 63.7 (m), 43.6 (t, J = 150.1 Hz), 15.2.31P NMR (81 MHz, MeOD-d4): d 16.1.MS (ESI+) m/z: 515.3 [M+H]+.

The final inhibitor (((5-phenylthieno[2,3-d]pyrimidin-4-yl)ami-no)methylene)diphosphonic acid (2c) was isolated as a white solidin 54% yield (5.1 mg)

1H NMR (500 MHz, D2O): d 8.17 (s, 1H), 7.68 (s, 1H), 7.54 (d,J = 7.0 Hz, 2H), 7.43 (t, J = 7.6 Hz, 2H), 7.35 (d, J = 7.4 Hz, 1H),7.12 (s, 1H).13C NMR (126 MHz, D2O): d 165.0, 153.2, 135.3, 129.4, 129.1,128.3, 120.7, 113.9, 50.6 (t, J = 123.6).31P NMR (81 MHz, D2O): d 13.4.HRMS (ESI�) Calcd 399.9928 for C13H12N3P2O6S, found m/z399.9918 [M�H]�.

4.7.5. (6-Phenylthieno[2,3-d]pyrimidin-4-ylamino)methylenebisphosphonic acid (2d)

The tetraethyl ((6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)-methylene bisphosphonate was isolated in 65% yield (38.0 mg).

1H NMR (500 MHz, CD3OD) d 8.45 (s, 1H), 8.04 (s, 1H), 7.75 (d,J = 7.3 Hz, 2H), 7.48 (t, J = 7.6 Hz, 2H), 7.41 (d, J = 7.3 Hz, 1H),6.00 (t, J = 23.6 Hz, 1H), 4.22 (dd, J = 7.7, 3.2 Hz, 8H), 1.45–1.09(m, 12H).13C NMR (126 MHz, CD3OD) d 167.0, 157.2, 154.1, 142.7, 134.6,130.4, 130.1, 127.2, 119.8, 115.2, 65.2, 45.6, 16.7.31P NMR (81 MHz, CD3OD) d 17.004.MS (ESI) m/z 536.15 [M+Na]+.

The final inhibitor (6-phenylthieno[2,3-d]pyrimidin-4-ylami-no)methylene bisphosphonic acid (2d) was isolated as a white so-lid in 63% yield (18.8 mg).

1H NMR (400 MHz, D2O, 10% ND4OD): d 8.18 (s, 1H), 7.80 (s,1H), 7.71 (d, J = 7.4 Hz, 2H), 7.42 (t, J = 7.6 Hz, 2H), 7.33 (t,J = 7.4 Hz, 1H).13C NMR (126 MHz, D2O, 10% ND4OD): d 163.1, 156.2, 153.6,139.2, 133.2, 129.3, 128.6, 125.9, 118.64, 114.8.31P NMR (81 MHz, D2O, ND4OD) d 13.29.HRMS (ESI�) Calcd 399.99166 for C13H12SN3O6P2, found399.99268 [M�H]�.

4.7.6. (((6-(p-Tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)diphosphonic acid (2e)

The tetraethyl (((6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)ami-no)methylene)bis(phosphonate) was isolated as a light brownpowder 94% (27.3 mg).

1H NMR (500 MHz, MeOD-d4): d 8.43 (s, 1H), 7.97 (s, 1H),7.60 (d, J = 8.1 Hz, 2H), 7.27 (d, J = 7.8 Hz, 1H), 6.00 (t,J = 23.6 Hz, 1H), 4.24–4.21 (m, 8H), 2.37 (s, 3H), 1.31–1.24(m, 12H).13C NMR (126 MHz, MeOD-d4): d 166.8, 157.0, 153.9, 142.9,140.4, 131.8, 130.9, 127.1, 119.8, 114.4, 65.10 (m), 45.6 (t,J = 150 Hz), 21.3, 16.7 (m).31P NMR (81 MHz, MeOD-d4): d 17.4.MS (ESI+) m/z: 528.2 [M+H]+.

The final inhibitor (((6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)a-mino)methylene)diphosphonic acid (2e) was isolated as a whitesolid in 94% yield (27.3 mg).

1H NMR (500 MHz, D2O): d 8.22 (s, 1H), 7.80 (s, 1H), 7.66 (d,J = 8.2 Hz, 2H), 7.30 (d, J = 8.0 Hz, 2H), 2.33 (s, 3H).

2240 C.-Y. Leung et al. / Bioorg. Med. Chem. 21 (2013) 2229–2240

13C NMR (126 MHz, D2O): d 164.3, 157.6, 155.0, 140.8, 140.7,131.9, 127.5, 120.2, 115.7, 21.8.31P NMR (81 MHz, D2O): d 13.72.HRMS (ESI�) Calcd 414.00840 for C14H14N3P2O6S, found m/z414.00863 [M�H]�.

4.7.7. (((6-(Naphthalen-2-yl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)diphosphonic acid (2f)

The tetraethyl (((6-(naphthalen-2-yl)thieno[2,3-d]pyrimidin-4-yl)amino)methylene)bis(phosphonate) was isolated as a whitepowder 71% (30.8 mg)

1H NMR (500 MHz, MeOD-d4): d 8.45 (s, 1H), 8.14 (s, 2H), 7.94–7.83 (m, 4H), 7.53–7.47 (m, 2H), 6.02 (t, J = 23.6 Hz, 1H), 4.24–4.21 (m, 8H), 1.31–1.24 (m, 12H).13C NMR (126 MHz, MeOD-d4): d 167.1, 157.1, 154.2, 142.6,135.0, 134.8, 131.9, 130.0, 129.3, 128.8, 128.0, 127.9, 126.3,124.6, 119.9, 115.6, 65.1 (m), 45.7 (t, J = 150 Hz), 16.7 (m).31P NMR (81 MHz, MeOD-d4): d 17.4.MS (ESI+) m/z: 564.3 [M+H]+.

The final inhibitor (((6-(naphthalen-2-yl)thieno[2,3-d]pyrimi-din-4-yl)amino)methylene)diphosphonic acid (2f) was isolated asa white solid in 86% yield (29.5 mg).

1H NMR (500 MHz, D2O): d 8.19 (s, 1H), 8.13 (s, 1H), 7.91–7.83(m, 5H), 7.50–7.44 (m, 2H).13C NMR (126 MHz, D2O): d 164.8, 157.8, 155.1, 140.7, 134.7,134.3, 132.2, 130.4, 129.6, 129.3, 128.6, 128.3, 126.2, 125.3,120.3, 116.8.31P NMR (81 MHz, D2O): d 13.74.HRMS (ESI�) Calcd 450.0084 for C17H14N3P2O6S, found m/z450.0085 [M�H]�.

Acknowledgments

Financial support for this work was provided by NSERC andFQRNT (research Grants to Y.S.T). A.L. is grateful to NSERC for grad-uate scholarship.

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bmc.2013.02.006.

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